1. Field of the Invention
This invention relates to a method of gene transfer, which enables stable expression of an exogenous gene in a plant cell.
2. Description of Related Art
Many kinds of transgenic plants with an exogenous gene introduced have been produced to render various characteristic to a plant. In the production of transgenic plants, the diversity of gene expression is observed among individual transgenic plants. It is considered that the diversity is caused by the position of introduced gene in a chromosome. When an exogenous gene is introduced into active chromatin region, high expression of the exogenous gene would be obtained. On the contrary, when an exogenous gene is introduced into inert chromatin region, sufficient expression of the gene would not be obtained (Galli, Current opinion in plant technology (1998) 1:166-172, Matzke et al., Current opinion in plant technology (1998) 1:142-148). Such effect described above is called "position effect". Because of the position effect, a transgene, which is introduced into a plant, exhibits absolute failure of expression, only weak expression or suspension of expression caused by plant growth or exogenous environment. This phenomenon serves as a barrier to commercialization of a transgenic plant and establishment of a method to stabilize expression of an introduced gene have been demanded.
Recently, some transformants, wherein genes of various kinds are introduced, revealed constant expression of the gene independent of its introduced position in the chromosome. The factors concerning such phenomenon are classified into three cases. These are, insulator, LCR (locus control region) and MAR (matrix attachment region) and involvement of such factors on constant expression is suggested. These three factors function as boundaries in a chromosome and blockade effects of near-existing chromatin, though the mechanism of action of these factors differs from each other.
When an insulator is located between an enhancer and a promoter, the insulator operates to blockade the effect of the enhancer as a silencer. In a higher eukaryotic plant, an enhancer and a silencer might cause an effect on transcriptional activity of not only a certain promoter, but also plural promoters. Therefore, plural elements might cause effects randomly in a chromosome. This phenomenon might work to render diversity on gene expression. On the other hand, a mechanism to restrict the enhancer function or the silencer function might be necessary for precise regulation of gene expression. At present, it is speculated that, an insulator might operate to restrict these functions.
LCR is a region higher-sensitive to DNasel. LCR might form active chromatin, which facilitates physical accession of promoter and various transcriptional factors.
MAR contains an adenine, thymine (AT) rich sequence and a topoisomerase II recognition sequence. Moreover, MAR exhibits nuclear-membrane binding activity under in virto condition. MAR is considered to exist more than every 10-100 kb in a chromosome, and the chromosome binds to nuclear membrane through these regions to form conformation of the chromosome. MAR is indispensable for compaction of chromosome in a limited space of nucleus. The knowledge indicating that the MAR might be involved in the regulation of gene expression is accumulating in recent researches. For example, the analysis of MAR derived from kappa light chain immunogloblin gene of mouse revealed following knowledge.
(1) MAR is necessary for gene expression in the process of development. PA0 (2) Inactivation of gene expression is caused by deletion of MAR through high-order methylation of DNA. PA0 (1) Plenty of MAR's might operate to increase expression of introduced gene. PA0 (2) When plural copies are introduced, expression of introduced gene might be suppressed by other effects than position effect, for example, specific methylation of DNA.
Moreover, a transcriptional unit of one or more gene is contained in a chromatin roop formed among plural MAR's. The analysis of beta globin gene wherein a cluster is formed in the loop revealed that, mutual affinity of each gene was indispensable for appropriate regulation and function of the gene. From these knowledge, it is speculated that, one chromatin loop among MAR's might form an independent unit, and gene expression might be regulated per the unit.
As described above, stable expression of introduced gene is demanded to obtain a practical transformant, regardless of whether the transformant is an animal or a plant. As described heretofore, avoidance of effect caused by surroundings of introduced location, a position effect, is indispensable. Therefore, the possibility that the position effect might be avoided by insertion of LCR, MAR or insulator have been investigated. In animal cell, LCR and insulator functioned as expected and the diversity of gene expression among transformants decreased (Akasaka, Cell Engineering (1997)16:1476-1484, Yasue et al. JP 6550/97). Concerning MAR, investigations performed by a promoter gene or a reporter gene did not exhibit consistent results. Therefore, stable expression of introduced gene was not achieved in many cases. In plants, this result might be caused by following phenomenon.
Therefore, MAR might not function as a boundary on the chromosome necessarily (Galli, Current opinion in plant technology (1998)1:166-172, Matzke et al., Current opinion in plant technology (1998)1:142-148), resulting in failure of generalization of the technique using MAR.